We propose to continue our studies on eukaryotic DNA replication, using DNA puffs from polytene chromosomes of the fly. Sciara coprophila, as a model system. DNA puffs are one of only two cases known to undergo intrachromosomal DNA amplification as a normal event in development (the other case is chorion gene amplification In Drosophila follicle cells). We have just mapped the origin for amplification to predominantly a l kb region In DNA puff 11/9A. Thus, it now provides the only non-viral, metazoan eukaryotic origin that has been mapped unambiguously to a small, well-defined region. We propose to continue our mapping studies to confirm this map location based on 2-D gels by using another method. We will complete our preliminary experiments that suggest that the same region is used as an origin for normal chromosomaI replication prior to amplification. We propose to develop techniques to map the origIn of replication to the nucleotide level; this technique will be developed using a well-defined ARS in yeast before application to Sciara DNA puffs. We will isolate and characterize genomic clones from another large DNA puff (11/2B). and map its origin. The origin regions in DNA puffs 11/9A and 1l/2B will be sequenced to allow comparison between them for Identification of conserved regions that could be of functional importance for replication. DNase I hypersensitivity studies will be completed, and in vivo and in vitro footprinting carried out to define areas that bind proteins in the origin region. The Importance of DNA sequences that function as the origin and the cis-acting elements that regulate origin activity will be tested by functional assays using P-element transformation. Cancer may be thought of as a disease of runaway DNA replication. so an understanding of the fundamental mechanism of DNA replication in eukaryotes and Its normal replication is key to future progress in cancer where the controls have seemingly gone awry.